小尾寒羊瘦素长型受体基因实时荧光定量PCR标准品质粒和标准曲线的构建

中国畜牧兽医

小尾寒羊瘦素长型受体基因实时荧光定量PCR标准品质粒和标准曲线的构建

张桂山¹，徐晶¹^，2，娄玉杰^{1，张善鹏¹，姜怀志¹}

（1.吉林农业大学动物科学技术学院，吉林长春 130118；2.长春科技学院，吉林长春 130600)

收稿日期:2013-09-25 出版日期:2014-03-20 发布日期:2014-05-15
通讯作者: 娄玉杰（1956—），女，吉林人，教授，博士生导师。E-mail：louyujie2003@aliyun.com
作者简介:张桂山（1978—），男，吉林人，博士生，研究方向：反刍动物营养与遗传。
基金资助:
吉林省科技支撑计划项目（20110237）。

Construction of Small-tailed Han Sheep Leptin Receptor Gene Standard Plasmid and Standard Curve Using Real-time Quantitative PCR

ZHANG Gui-shan¹, XU Jing¹^{, 2}, LOU Yu-jie¹, ZHANG Shan-peng¹, JIANG Huai-zhi¹

.（1.College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, China；2.Changchun University of Science and Technology, Changchun 130600, China）

Received:2013-09-25 Online:2014-03-20 Published:2014-05-15

摘要/Abstract

摘要： 本试验旨在制备用于小尾寒羊瘦素长型受体（leptin receptor long form）基因实时荧光定量PCR的标准品质粒和标准曲线。提取初情期前小尾寒羊下丘脑弓状核组织总RNA，进行反转录合成第1链cDNA，然后进行PCR和目的基因片段的回收；通过T-A克隆将该基因片段插入pMD18-T载体中，构建回收产物的重组质粒；大量提取质粒，定量后进行标准曲线的制作和实时荧光定量PCR。重组质粒经PCR扩增和测序，结果表明，瘦素长型受体基因已成功克隆。提示，所构建的瘦素长型受体基因实时荧光定量标准曲线线性关系好，灵敏度和特异性高，准确可靠，此方法可作为实时荧光定量PCR检测瘦素长型受体基因的标准方法。

关键词: 瘦素长型受体; 实时荧光定量PCR; 标准品质粒

Abstract: In order to prepare the leptin receptor long form gene standard plasmid and standard curve using Real-time quantitative PCR, the total RNA was isolated from the arcuate nucleus tissue of ovis aries before puberty, and was synthesized the first-strand cDNA via reverse transcription.Using PCR technology, the leptin receptor long form gene was amplified.The aim gene fragment was recycled, and the fragment was then cloned into the pMD18-T plasmid vector.Recombinant plasmid of recycled products was constructed.After extraction and identification, the recombinant plasmids were quantified and the standard curve was established.The amplified products of the recombinant plasmids and secquence analysis confirmed that the pMD18-T-leptin receptor long form gene was successfully cloned.The standard curve showed high linearity, sensitivity, specificity and exercisable, which can be used to detect leptin receptor long form gene expression

Key words: leptin receptor long form; Real-time quantitative PCR; standard plasmid

张桂山，徐晶，娄玉杰，张善鹏，姜怀志. 小尾寒羊瘦素长型受体基因实时荧光定量PCR标准品质粒和标准曲线的构建[J]. 中国畜牧兽医.

ZHANG Gui-shan, XU Jing, LOU Yu-jie, ZHANG Shan-peng, JIANG Huai-zhi. Construction of Small-tailed Han Sheep Leptin Receptor Gene Standard Plasmid and Standard Curve Using Real-time Quantitative PCR[J]. .

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